Hi, my name is Marianne and I’m going to be a senior next year at Lynbrook High School in San Jose, California. I’ve lived in California all my life, though I used to live near Los Angeles when I was younger. I’ve always been interested in biology, and last year, after taking AP Biology, I decided that I wanted to pursue it as a career. I really enjoyed learning about all the topics and labs were so much fun to do, even though we didn’t have many opportunities for them. I chose this program because I wanted to take my interest further and learn some lab techniques that will be helpful later on. I’m also really looking forward to creating my research project so I can have the experience while learning about modern-day applications. I want to pursue a job in medicine, though I’m not yet sure what specifically. I love doing colorguard, which takes up most of my time, but I’m also very active in school clubs such as CSF and Red Cross. Along with the community service for those clubs, I volunteer at a local hospital.
Histoplasmosis is a fungal infection that is very widespread and affects more than 1 million people worldwide. It is the most common of three major fungal infections endemic to North America. People are usually infected by inhaling fungal spores of the species Histoplasma capsulatum, commonly found in bird and bat droppings. These spores are also distributed in soils all over the world, though cases are extremely common around the Ohio and Mississippi River valleys, where around 90% of people are positive for histoplasmin skin tests. It is also common in Latin America and Africa.
The main issue with histoplasmosis arises in HIV patients, who have weakened immune systems and are extremely vulnerable to bacterial and fungal infections. The Centers for Disease Control and Prevention has named histoplasmosis to be an AIDS-defining illness, meaning that an HIV patient infected with it has developed AIDS. The disease is more dangerous in poorer countries where antiretroviral therapy, or ART, is not as readily available. ART is used to significantly reduce total HIV DNA levels, which can slow the disease and help prevent patients from reaching vulnerable states. For example, in Latin America, almost 30% of HIV patients that are diagnosed with histoplasmosis die from it.
Histoplasmosis mainly occurs in the lungs, though it has the potential to spread throughout the rest of the body. Most people do not experience symptoms even after exposure to the fungus, but some will experience mild flu-like symptoms for a short span. These include fever, swollen lymph glands, cough, shortness of breath, and severe weight loss. For HIV patients, histoplasmosis can lead to a chronic lung infection similar to tuberculosis that, if untreated, leads to enlarged lung cavities and loss of lung function. If the infection spreads throughout the body, it becomes “disseminated disease” and is extremely severe and even life-threatening. It can affect multiple organs, and some of the worse symptoms are enlarged spleen and liver, gastrointestinal bleeding, and bone marrow failure.
Many acute histoplasmosis cases are mild and resolve themselves after a few weeks without treatment. Chronic histoplasmosis can be treated with antifungals for a year until symptoms disappear. Disseminated histoplasmosis must be treated with a combination of oral drugs with an IV; however, the underlying immunosuppression must be treated for the disease to fully resolve itself.
The fungus H. capsulatum is thermally dimorphic and grows as mycelium in the environment. However, at body temperature, which is around 37℃, it turns into yeast. The main form of the fungus is microconidia, which are asexual, non-motile spores. These microconidia are inhaled into the alveoli, where macrophages consume them. However, the spores survive and, because of the change in temperature, transform into yeast which multiply in the phagosome. As the macrophages circulate in the lymphatic system, the fungus is spread to organs around the body. While inside the phagosome, H. capsulatum has an absolute requirement for thiamine.
Design: My proposal is a synthetically engineered bacteria that can prevent the germination of fungal spores. To achieve this, we must consider the process of conidial germination. It is when a conidium, or spore, forms germ tubes that grow to become the hyphae of a developed fungus.
One experiment was conducted to study the effects of various alternative chemicals on the inhibition of mycelium growth and conidia germination of one fungus, B. cinerea. We will focus specifically on the effects of conidia germination. Ipradion, a standard fungicide, was used for comparison to see how effective the chemicals were.
Isolate-1: From the results of the experiment, we can see that sorbic acid at a dose of 30 μg mL-1 inhibited conidia germination considerably in comparison to the other chemicals. Looking at the grander scheme, ipradion was the only chemical to effectively inhibit germination at low doses (around 3 μg mL-1) while other alternative chemicals failed. However, as ipradion is an actual fungicide, we must disregard it and look at the next best option, which in this experiment was sorbic acid. The next lowest concentration among the alternative chemicals was sodium benzoate at 100 μg mL-1, a much greater amount than the 30 μg mL-1 of sorbic acid. In addition, both compounds completely inhibited conidial germination at a concentration of 300 μg mL-1.
table 3.2.png
Isolate-2: In another isolate, sorbic acid had the lowest minimum inhibition concentration on conidia germination of Isolate-2 after ipradion, which verifies its effectiveness in two scenarios.
table 5.png
Table 5 also demonstrates how, after ipradion, sorbic acid had the lowest ED50 values on conidial germination of both isolates.
With the experiment in mind, my design aims to produce sorbic acid in the human body as a way to prevent fungal spores of H. capsulatum from germinating and developing into yeast, which would eventually lead to histoplasmosis. Sorbic acid is naturally produced in berries of Sorbus aucuparia, or the rowan tree. Though the specific gene has not yet been identified, there is potential for scientists to identify it and insert it into bacteria. For my design, I plan to use E. coli, a naturally occurring bacteria in the intestines, as the chassis for the gene. In the presence of specific chemicals released by H. capsulatum conidia, the gene would be activated and would produce an enzyme that could manufacture sorbic acid. From the intestines, the sorbic acid would be absorbed into the bloodstream and would eventually make its way to the lungs, the primary site of infection for H. capsulatum. There, the chemical could prevent fungal spores from germinating, thus reducing the risk of getting histoplasmosis.
Screen Shot 2016-08-04 at 12.12.21 PM.png
Expected results:
H. capsulatum Conidia present
Sorbic Acid present
H. capsulatum Conidial Germination
1
1
0
0
0
1
Advantages: The bacteria acts mainly as a preventative measure. Since the E. coli would inhibit conidial germination, it would theoretically prevent most or any development of the fungus H. capsulatum, thereby preventing risk of infection or further complications caused by histoplasmosis. In areas such as the Ohio and Mississippi River valleys, HIV patients who are greatly at risk for infection by H. capsulatum can use this treatment as a precaution. The main advantage of this design is the use of sorbic acid, a natural organic compound. It is widely used as a food preservative so it has already been approved for human ingestion. Sorbic acid has been often chosen as a food preservative over nitrates, which form carcinogenic byproducts in the human body, as compared to its relative harmlessness. In addition, the compound is relatively unstable so it rapidly degrades in soil and is environmentally friendly.
Potential Problems: Some potential problems with the design is that sometimes sorbic acid can cause allergic contact dermatitis. However, this is quite rare, and many patients can simply reduce symptoms by drinking 8 ounces of water. One issue that may reduce effectiveness of this design is that sorbic acid may take some time to circulate in the blood from the intestines, where E. coli would produce it, to the lungs where the majority of fungal spores are found. Also, with the use of any bacteria there is the risk of infection for workers, but this can be reduced by using harmless strains and by taking necessary precautions.
Testing: To test the design, one would have to ensure the effectiveness of sorbic acid on conidial germination of H. capsulatum. This can be done by creating isolates in vitro and comparing results with traditional fungicides, similar to the experiment mentioned above. If conidial germination is significantly reduced, it would ensure that sorbic acid works. This design also has future potential applications for other fungal infections.
Histoplasmosis is a fungal infection that is very widespread and affects more than 1 million people worldwide. It is the most common of three major fungal infections endemic to North America. People are usually infected by inhaling fungal spores of the species Histoplasma capsulatum, commonly found in bird and bat droppings. These spores are also distributed in soils all over the world, though cases are extremely common around the Ohio and Mississippi River valleys, where around 90% of people are positive for histoplasmin skin tests. It is also common in Latin America and Africa.
The main issue with histoplasmosis arises in HIV patients, who have weakened immune systems and are extremely vulnerable to bacterial and fungal infections. The Centers for Disease Control and Prevention has named histoplasmosis to be an AIDS-defining illness, meaning that an HIV patient infected with it has developed AIDS. The disease is more dangerous in poorer countries where antiretroviral therapy, or ART, is not as readily available. ART is used to significantly reduce total HIV DNA levels, which can slow the disease and help prevent patients from reaching vulnerable states. For example, in Latin America, almost 30% of HIV patients that are diagnosed with histoplasmosis die from it.
Histoplasmosis mainly occurs in the lungs, though it has the potential to spread throughout the rest of the body. Most people do not experience symptoms even after exposure to the fungus, but some will experience mild flu-like symptoms for a short span. These include fever, swollen lymph glands, cough, shortness of breath, and severe weight loss. For HIV patients, histoplasmosis can lead to a chronic lung infection similar to tuberculosis that, if untreated, leads to enlarged lung cavities and loss of lung function. If the infection spreads throughout the body, it becomes “disseminated disease” and is extremely severe and even life-threatening. It can affect multiple organs, and some of the worse symptoms are enlarged spleen and liver, gastrointestinal bleeding, and bone marrow failure.
Many acute histoplasmosis cases are mild and resolve themselves after a few weeks without treatment. Chronic histoplasmosis can be treated with antifungals for a year until symptoms disappear. Disseminated histoplasmosis must be treated with a combination of oral drugs with an IV; however, the underlying immunosuppression must be treated for the disease to fully resolve itself.
The fungus H. capsulatum is thermally dimorphic and grows as mycelium in the environment. However, at body temperature, which is around 37℃, it turns into yeast. The main form of the fungus is microconidia, which are asexual, non-motile spores. These microconidia are inhaled into the alveoli, where macrophages consume them. However, the spores survive and, because of the change in temperature, transform into yeast which multiply in the phagosome. As the macrophages circulate in the lymphatic system, the fungus is spread to organs around the body. While inside the phagosome, H. capsulatum has an absolute requirement for thiamine.
Design:
My proposal is a synthetically engineered bacteria that can prevent the germination of fungal spores. To achieve this, we must consider the process of conidial germination. It is when a conidium, or spore, forms germ tubes that grow to become the hyphae of a developed fungus.
One experiment was conducted to study the effects of various alternative chemicals on the inhibition of mycelium growth and conidia germination of one fungus, B. cinerea. We will focus specifically on the effects of conidia germination. Ipradion, a standard fungicide, was used for comparison to see how effective the chemicals were.
Isolate-1:
From the results of the experiment, we can see that sorbic acid at a dose of 30 μg mL-1 inhibited conidia germination considerably in comparison to the other chemicals. Looking at the grander scheme, ipradion was the only chemical to effectively inhibit germination at low doses (around 3 μg mL-1) while other alternative chemicals failed. However, as ipradion is an actual fungicide, we must disregard it and look at the next best option, which in this experiment was sorbic acid. The next lowest concentration among the alternative chemicals was sodium benzoate at 100 μg mL-1, a much greater amount than the 30 μg mL-1 of sorbic acid. In addition, both compounds completely inhibited conidial germination at a concentration of 300 μg mL-1.
Isolate-2:
In another isolate, sorbic acid had the lowest minimum inhibition concentration on conidia germination of Isolate-2 after ipradion, which verifies its effectiveness in two scenarios.
Table 5 also demonstrates how, after ipradion, sorbic acid had the lowest ED50 values on conidial germination of both isolates.
With the experiment in mind, my design aims to produce sorbic acid in the human body as a way to prevent fungal spores of H. capsulatum from germinating and developing into yeast, which would eventually lead to histoplasmosis. Sorbic acid is naturally produced in berries of Sorbus aucuparia, or the rowan tree. Though the specific gene has not yet been identified, there is potential for scientists to identify it and insert it into bacteria. For my design, I plan to use E. coli, a naturally occurring bacteria in the intestines, as the chassis for the gene. In the presence of specific chemicals released by H. capsulatum conidia, the gene would be activated and would produce an enzyme that could manufacture sorbic acid. From the intestines, the sorbic acid would be absorbed into the bloodstream and would eventually make its way to the lungs, the primary site of infection for H. capsulatum. There, the chemical could prevent fungal spores from germinating, thus reducing the risk of getting histoplasmosis.
Expected results:
Advantages:
The bacteria acts mainly as a preventative measure. Since the E. coli would inhibit conidial germination, it would theoretically prevent most or any development of the fungus H. capsulatum, thereby preventing risk of infection or further complications caused by histoplasmosis. In areas such as the Ohio and Mississippi River valleys, HIV patients who are greatly at risk for infection by H. capsulatum can use this treatment as a precaution. The main advantage of this design is the use of sorbic acid, a natural organic compound. It is widely used as a food preservative so it has already been approved for human ingestion. Sorbic acid has been often chosen as a food preservative over nitrates, which form carcinogenic byproducts in the human body, as compared to its relative harmlessness. In addition, the compound is relatively unstable so it rapidly degrades in soil and is environmentally friendly.
Potential Problems:
Some potential problems with the design is that sometimes sorbic acid can cause allergic contact dermatitis. However, this is quite rare, and many patients can simply reduce symptoms by drinking 8 ounces of water. One issue that may reduce effectiveness of this design is that sorbic acid may take some time to circulate in the blood from the intestines, where E. coli would produce it, to the lungs where the majority of fungal spores are found. Also, with the use of any bacteria there is the risk of infection for workers, but this can be reduced by using harmless strains and by taking necessary precautions.
Testing:
To test the design, one would have to ensure the effectiveness of sorbic acid on conidial germination of H. capsulatum. This can be done by creating isolates in vitro and comparing results with traditional fungicides, similar to the experiment mentioned above. If conidial germination is significantly reduced, it would ensure that sorbic acid works. This design also has future potential applications for other fungal infections.
Bibliography:
__https://www.verywell.com/opportunistic-fungal-infections-47932__
__https://www.verywell.com/what-are-the-aids-defining-illnesses-49581__
__http://www.lung.org/lung-health-and-diseases/lung-disease-lookup/histoplasmosis/__
__http://medicalxpress.com/news/2016-08-early-antiretroviral-therapy-hiv-dna.html__
__http://www.lung.org/lung-health-and-diseases/lung-disease-lookup/histoplasmosis/diagnosing-and-treating.html__
__https://www.dhs.wisconsin.gov/publications/p4/p42058.pdf__
__http://emedicine.medscape.com/article/299054-overview#showall__
__http://www.merckmanuals.com/home/infections/fungal-infections/histoplasmosis__
__http://www.cdc.gov/fungal/diseases/histoplasmosis/index.html__
__http://www.healthline.com/health/food-nutrition/what-is-sorbic-acid__
http://scialert.net/fulltext/?doi=pjbs.2007.1294.1300#t2
Images:
__http://www.cdc.gov/fungal/images/histoplasmosis-lifecycle-maps-300px.jpg__
__http://www.avert.org/professionals/hiv-science/overview__
__http://dxline.info/img/new_ail/histoplasmosis-acute-pulmonary_1.jpg__
__http://media.npr.org/assets/img/2013/10/24/histo-64379ac1c677ff1c85cc501c9b7965a48bd5c59f-s6-c30.jpg__
__http://botit.botany.wisc.edu/toms_fungi/images/hcapyeast.jpg__
__http://faculty.ccbcmd.edu/courses/bio141/labmanua/lab10/images/u1fig39b.gif__
__https://classconnection.s3.amazonaws.com/497/flashcards/3433497/jpg/histoplasmosis_life_cycle-13FF8C6B25051069473.jpg__
__http://www.peoi.org/Courses/Coursesen/bot/Resources/image1413.jpg__